Eugene S. Baginski

Determination of phosphate: Study of labile organic phosphate interference☆

Abstract An investigation was carried out on a newer method for the determination of inorganic phosphate which is applicable to the study of phosphate-splitting enzymes. It offers a unique advantage: after the color reagent has been added any inorganic phosphate formed cannot react with molybdenum, for the latter has been complexed by the addition of a citrate-arsenite solution and phosphate cannot compete successfully for the molybdate. This procedure appears to be quite suitable for the determination of inorganic phosphate, phospholipids, nucleotides or enzyme-hydrolyzed phosphate.

Glucose-6-phosphatase

Publisher Summary This chapter focuses on glucose-6-phosphatase (G6Pase), which was first described by Fantl et al. Numerous attempts have been made to determine G6Pase activity in blood and so relate its activity to liver damage. G6Pase is applied in biochemistry and clinical chemistry. The inorganic phosphate liberated is determined with ammonium molybdate; ascorbic acid is used as the reducing agent. The amount of phosphate liberated per unit time, determined as the blue phosphomolybdous complex at 700 or 840 nm, is a measure of the G6Pase activity. The optimum pH for the enzyme reaction lies between 6.2 and 6.51. Only doubly distilled water should be used. The sucrose, G-6-P solution and the cacodylate buffer should be stored at 0–4°C. Freshly obtained tissue should be homogenized first in a precooled blender and then in glass-Teflon homogenizer with ice-cold sucrose solution. The enzyme is stable for several months at −35°C in the absence of EDTA. The instability of the enzyme causes serious errors, it is therefore necessary to assay the suspensions immediately after thawing. The enzyme is very specific for G-6-P, although other phosphate esters can be hydrolyzed.

Determination of rat liver microsomal glucose-6-phosphatase activity: Study of citrate and G-6-P inhibition

Abstract Experiments were carried out to study the effect of citrate, G-6-P concentration, and pH on the G-6-Pase activity of liver microsomes. It was shown that, under the described experimental conditions, the following hold: 1. 1. Citrate ion neither inhibited nor enhanced G-6-Pase activity, and did not interfere with the determination of inorganic phosphate. 2. 2. Cacodylate buffer was more suitable for these enzymes studies than citrate buffer, but both buffers could be omitted from the incubation mixture if the pH of G-6-P substrate is preadjusted to 6.2. 3. 3. Maximum enzyme activity was obtained between G-6-P concentration of 20 and 50 μmoles present in 0.4 ml of reaction mixture at a pH between 6.2 and 6.5. 4. 4. The degree of nonenzymic acid hydrolysis of G-6-P was insignificant.

The direct weighing of ziorconium tetramandelate

Abstract The non-stoichiometry of zirconium tetramandelate precipitates is explained by the presence of basic salts of varying composition. For reproducible results the zirconium tetramandelate should be precipitated from hot. strongly acid solution by the dropwise addition of mandelic acid.

AUTOMATION OF METHODS FOR THE DETERMINATION OF SERUM PHOSPHORUS.

Abstract Three procedures for the automated determination of serum inorganic phosphate have been described. For each of these technics, an accurate manual variation is available when necessary. A set of recoveries was made to indicate the comparable nature of the three automated procedures. A second set of recoveries showed that the two manual procedures proposed yielded results which were similar to presently acceptable methods, and that one of the three comparable automated technics is equally as accurate as the manual technic. This interchangeable technic of comparison of the three automated and manual procedures to each other and to acceptable manual methods shows that the proposed technics are analytically acceptable.

2,6-Bis(4-phenyl-2-pyridyl)-4-phenyl-pyridine as an aqueous micro iron reagent☆

Abstract A procedure for the determination of microquantities of iron by the use of water solubilized terosite was described. Some of the parameters of the procedure were discussed along with comparative spectra and recoveries. The study of the application of this procedure to human serum analysis for iron is presently in progress.

Microdetermination of nucleic acid phosphate

Abstract This paper describes a simple procedure for the determination of ribonucleic acid-deoxyribonucleic acid phosphate. The destruction step is rapid, the color reaction sensitive, and the process reproducible and accurate. The final color is stable, a phenomenon made possible by masking the excess molybdate by a citrate-arsenite mixture. Since there is no available molybdate after the masking step occurs, no color can form after that produced by the phosphate originally present.

Direct serum inorganic phosphate determination

Abstract A simple serum microinorganic phosphate determination is described which obviates protein precipitation. Chylous or turbid specimens can also be processed, but a sample blank must be included. The latter treatment is simple and is required only infrequently. Two sets of specimens were investigated for their inorganic phosphate content. One set included clear or slightly opalescent serums while the other set consisted of chylous and turbid specimens. Both sets were analyzed for phosphate content by the new direct technique with the sample correction applied to only the second set. In addition, phosphate concentration was determined in all specimens from both sets using a protein precipitation technique considered here as a reference procedure (3). The results obtained for both sets by the direct technique correlated well with those obtained by the reference method. Jaundiced specimens do not present a problem because the yellow color due to bilirubin does not absorb at the wavelength used. The red hemoglobin color does not interfere for the same reason. However, grossly hemolyzed specimens are not suitable because of the high phosphate content in erythrocytes.

Accelerated automated microdetermination of serum calcium

Abstract A rapid procedure for the automated determination of serum calcium by direct determination in raw serum is described. In this case, a 50/ hour direct serum determination was modified to operate at 100/hour, but the increased rate of processing did not affect accuracy, reproducibility of the determinations or sample to sample interaction. Dimethyl sulfoxide was included in the reagents as a solvent to lower the dielectric constant of the medium and to help repress the ionization of the color reagent, cresolphthalein complexone, thereby decreasing the blank. Potential interference from lipemia, jaundice or hemolysis was not covered in the study because the primary stated purpose here was to develop a model concept for accelerated on-stream automation. However, further studies along these lines are in progress. A discrete sample analyzer, the Beckman DSA 560 was also used for the determination and the same reagents were adequate. Acceleration of direct automated determinations such as described seems feasible within limits when using the concept of manifold lines enlarged in the ratio of desired faster to slower speeds. Recoveries indicate that the proposed accelerated model system is useful for the determination of serum calcium.

A solvent problem in barbiturate determinations

Abstract Two common problems in the quantitative determination of serum barbiturate are described, and methods of elimination of errors engendered by them are suggested. A substance present in the chloroform used to extract serum for barbiturate distorts the difference spectra graphed. This interferes with both qualitative identification of the therapeutic barbiturate class as well as the concentration of barbiturate found. A simple wash of the chloroform by the same alkali used in the back-extraction process suffices to remove this interference. Testing the reagents, which really only represents a reagent blank, quickly identifies whether a reagent problem exists. Another problem arises owing to the normally alkaline pH of serum at room temperature. It can be solved preferably by acidification, or else the ratio of extractant volume to serum volume should be increased, or two extractions of the sample must be carried out (7).